UDP-glucose dehydrogenase (UGDH) activity is suppressed by peroxide and promoted by PDGF in fibroblast-like synoviocytes: Evidence of a redox control mechanism.

UDP-glucose dehydrogenase (UGDH) generates essential precursors of hyaluronic acid (HA) synthesis, however mechanisms regulating its activity are unclear. We used enzyme histostaining and quantitative image analysis to test whether cytokines that stimulate HA synthesis upregulate UGDH activity. Fibroblast-like synoviocytes (FLS, from N = 6 human donors with knee pain) were cultured, freeze-thawed, and incubated for 1 hour with UDP-glucose, NAD+ and nitroblue tetrazolium (NBT) which allows UGDH to generate NADH, and NADH to reduce NBT to a blue stain. Compared to serum-free medium, FLS treated with PDGF showed 3-fold higher UGDH activity and 6-fold higher HA release, but IL-1beta/TGF-beta1 induced 27-fold higher HA release without enhancing UGDH activity. In selected proliferating cells, UGDH activity was lost in the cytosol, but preserved in the nucleus. Cell-free assays led us to discover that diaphorase, a cytosolic enzyme, or glutathione reductase, a nuclear enzyme, was necessary and sufficient for NADH to reduce NBT to a blue formazan dye in a 1-hour timeframe. Primary synovial fibroblasts and transformed A549 fibroblasts showed constitutive diaphorase/GR staining activity that varied according to supplied NADH levels, with relatively stronger UGDH and diaphorase activity in A549 cells. Unilateral knee injury in New Zealand White rabbits (N = 3) stimulated a coordinated increase in synovial membrane UGDH and diaphorase activity, but higher synovial fluid HA in only 2 out of 3 injured joints. UGDH activity (but not diaphorase) was abolished by N-ethyl maleimide, and inhibited by peroxide or UDP-xylose. Our results do not support the hypothesis that UGDH is a rate-liming enzyme for HA synthesis under catabolic inflammatory conditions that can oxidize and inactivate the UGDH active site cysteine. Our novel data suggest a model where UGDH activity is controlled by a redox switch, where intracellular peroxide inactivates, and high glutathione and diaphorase promote UGDH activity by maintaining the active site cysteine in a reduced state, and by recycling NAD+ from NADH.

Dysregulated glucuronic acid metabolism exacerbates hepatocellular carcinoma progression and metastasis through the TGFß signalling pathway.

BACKGROUND: Glucuronic acid metabolism participates in cellular detoxification, extracellular matrix remodeling and cell adhesion and migration. Here, we aimed to explore the crosstalk between dysregulated glucuronic acid metabolism and crucial metastatic signalling in glutathione S-transferase zeta 1 (GSTZ1)-deficient hepatocellular carcinoma (HCC). METHODS: Transwell, HCC xenograft and Gstz1-/- mouse models were used to examine the role of GSTZ1 in HCC metastasis. Non-targeted and targeted metabolomics and global transcriptomic analyses were performed to screen significantly altered metabolic and signalling pathways in GSTZ1 overexpressing hepatoma cells. Further, RNA-binding protein immunoprecipitation, Biotin-RNA pull-down, mRNA decay assays and luciferase reporter assays were used to explore the interaction between RNA and RNA-binding proteins. RESULTS: GSTZ1 was universally silenced in both human and murine HCC cells, and its deficiency contributed to HCC metastasis in vitro and in vivo. UDP-glucose 6-dehydrogenase (UGDH)-mediated UDP-glucuronic acid (UDP-GlcUA) accumulation promoted hepatoma cell migration upon GSTZ1 loss. UDP-GlcUA stabilized TGFßR1 mRNA by enhancing its binding to polypyrimidine tract binding protein 3, contributing to the activation of TGFß/Smad signalling. UGDH or TGFßR1 blockade impaired HCC metastasis. In addition, UGDH up-regulation and UDP-GlcUA accumulation correlated with increased metastatic potential and decreased patient survival in GSTZ1-deficient HCC. CONCLUSIONS: GSTZ1 deficiency and subsequent up-regulation of the glucuronic acid metabolic pathway promotes HCC metastasis by increasing the stability of TGFßR1 mRNA and activating TGFß/Smad signalling. UGDH and a key metabolite, UDP-GlcUA, may serve as prognostic markers. Targeting UGDH might be a promising strategy for HCC therapy.

UDP-glucose 6-dehydrogenase lessens sorafenib sensitivity via modulating unfolded protein response.

As the first-generation targeted therapy, sorafenib remains an effective single-drug treatment for advanced hepatocellular carcinoma (HCC). Unfortunately, the existence of resistance restricts the long-term benefit of patients. UDP-glucose 6-dehydrogenase (UGDH) is the key enzyme of glucuronic acid metabolism which was largely reported in mediating drug systemic elimination. In this study, we explore its critical role in regulating sorafenib sensitivity. Here we find sorafenib exposure could activate glucuronic acid metabolism, accompanied with the elevated expression of UGDH. Interference with the route by silencing UGDH could boost HCC cells sensitivity to sorafenib. Meanwhile, the analysis of HCC patients with sorafenib treatment displayed that low UGDH expression predicted superior prognosis. Further screening assay suggested that unfolded protein response (UPR) involves in UGDH silencing-mediated apoptosis. Xenograft model confirmed that combined UGDH intervention could significantly improve sorafenib efficacy. Our results reveal the impact of sorafenib exposure on glucuronic acid metabolism reprogramming and provide UGDH as a promising target to improve sorafenib efficacy.

UDP-glucose dehydrogenase expression is upregulated following EMT and differentially affects intracellular glycerophosphocholine and acetylaspartate levels in breast mesenchymal cell lines.

Metabolic rewiring is one of the indispensable drivers of epithelial-mesenchymal transition (EMT) involved in breast cancer metastasis. In this study, we explored the metabolic changes during spontaneous EMT in three separately established breast EMT cell models using a proteomic approach supported by metabolomic analysis. We identified common proteomic changes, including the expression of CDH1, CDH2, VIM, LGALS1, SERPINE1, PKP3, ATP2A2, JUP, MTCH2, RPL26L1 and PLOD2. Consistently altered metabolic enzymes included the following: FDFT1, SORD, TSTA3 and UDP-glucose dehydrogenase (UGDH). Of these, UGDH was most prominently altered and has previously been associated with breast cancer patient survival. siRNA-mediated knock-down of UGDH resulted in delayed cell proliferation and dampened invasive potential of mesenchymal cells and downregulated expression of the EMT transcription factor SNAI1. Metabolomic analysis revealed that siRNA-mediated knock-down of UGDH decreased intracellular glycerophosphocholine (GPC), whereas levels of acetylaspartate (NAA) increased. Finally, our data suggested that platelet-derived growth factor receptor beta (PDGFRB) signalling was activated in mesenchymal cells. siRNA-mediated knock-down of PDGFRB downregulated UGDH expression, potentially via NFkB-p65. Our results support an unexplored relationship between UGDH and GPC, both of which have previously been independently associated with breast cancer progression.

Functional and Structural Characterization of the UDP-Glucose Dehydrogenase Involved in Capsular Polysaccharide Biosynthesis from Campylobacter jejuni.

Campylobacter jejuni is a pathogenic organism that can cause campylobacteriosis in children and adults. Most commonly, campylobacter infection is brought on by consumption of raw or undercooked poultry, unsanitary drinking water, or pet feces. Surrounding the C. jejuni bacterium is a coat of sugar molecules known as the capsular polysaccharide (CPS). The capsular polysaccharide can be very diverse among the different strains of C. jejuni, and this diversity is considered important for evading the host immune system. Modifications to the CPS of C. jejuni NCTC 11168 include O-methylation, phosphoramidylation, and amidation of glucuronate with either serinol or ethanolamine. The enzymes responsible for amidation of glucuronate are currently unknown. In this study, Cj1441, an enzyme expressed from the CPS biosynthetic gene cluster in C. jejuni NCTC 11168, was shown to catalyze the oxidation of UDP-α-d-glucose into UDP-α-d-glucuronic acid with NAD+ as the cofactor. No amide products were found in an attempt to determine whether the putative thioester intermediate formed during the oxidation of UDP-glucose by Cj1441 could be captured in the presence of added amines. The three-dimensional crystal structure of Cj1441 was determined in the presence of NAD+ and UDP-glucose bound in the active site of the enzyme (Protein Data Bank entry 7KWS). A more thorough bioinformatic analysis of the CPS gene cluster suggests that the amidation activity is localized to the t-terminal half of Cj1438, a bifunctional enzyme that is currently annotated as a sugar transferase.

Initial Identification of UDP-Glucose Dehydrogenase as a Prognostic Marker in Breast Cancer Patients, Which Facilitates Epirubicin Resistance and Regulates Hyaluronan Synthesis in MDA-MB-231 Cells.

UDP-glucose-dehydrogenase (UGDH) synthesizes UDP-glucuronic acid. It is involved in epirubicin detoxification and hyaluronan synthesis. This work aimed to evaluate the effect of UGDH knockdown on epirubicin response and hyaluronan metabolism in MDA-MB-231 breast cancer cells. Additionally, the aim was to determine UGDH as a possible prognosis marker in breast cancer. We studied UGDH expression in tumors and adjacent tissue from breast cancer patients. The prognostic value of UGDH was studied using a public Kaplan-Meier plotter. MDA-MB-231 cells were knocked-down for UGDH and treated with epirubicin. Epirubicin-accumulation and apoptosis were analyzed by flow cytometry. Hyaluronan-coated matrix and metabolism were determined. Autophagic-LC3-II was studied by Western blot and confocal microscopy. Epirubicin accumulation increased and apoptosis decreased during UGDH knockdown. Hyaluronan-coated matrix increased and a positive modulation of autophagy was detected. Higher levels of UGDH were correlated with worse prognosis in triple-negative breast cancer patients that received chemotherapy. High expression of UGDH was found in tumoral tissue from HER2--patients. However, UGDH knockdown contributes to epirubicin resistance, which might be associated with increases in the expression, deposition and catabolism of hyaluronan. The results obtained allowed us to propose UGDH as a new prognostic marker in breast cancer, positively associated with development of epirubicin resistance and modulation of extracellular matrix.

Exciting New Developments and Emerging Themes in Glycosaminoglycan Research.

In times where many people have suffered loss and others of us are dealing with stress, disruption, and fear, there is a lot of comfort to be taken in reading. If we are not able to meet up and discuss our work in person, exploring published studies provides some succor, even without the cheese, wine, and other traditions of our usual get-togethers. Fortunately, recent months have seen many high-quality papers around the topic of glycosaminoglycans. I can only pick up on a very few here, those that I have particularly enjoyed, but the following collection of reviews will also be a treat and hopefully tide us over until our research community can regroup.

Slit2 Regulates Hyaluronan & Cytokine Synthesis in Fibrocytes: Potential Relevance to Thyroid-Associated Ophthalmopathy.

CONTEXT: CD34+ fibrocytes have been implicated in development of thyroid-associated ophthalmopathy (TAO), a consequential autoimmune manifestation of Graves disease (GD). In TAO, CD34+ fibrocytes appear to masquerade as CD34+ orbital fibroblasts mixed with CD34- OF (collectively, GD-OF). Slit2, an axon guidance glycoprotein, is expressed by CD34- OF and attenuates GD-OF gene expression. Cardinal features of TAO include hyaluronan (HA) accumulation and cytokine-driven inflammation. OBJECTIVE: Compare expression of HA synthase isoenzymes (HAS1-3), UDP-glucose dehydrogenase (UGDH), synthesis of HA, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in fibrocytes and GD-OF. Determine whether Slit2 alters gene expression patterns. DESIGN/SETTING/PARTICIPANTS: Patients with TAO and healthy donors were recruited from an academic practice. MAIN OUTCOME MEASURES: Real-time polymerase chain reaction, HA, IL-6, and TNF-α immunoassays. RESULTS: HA synthesis and release from fibrocytes is substantially lower than in GD-OF. HAS1 expression dominates in fibrocytes while HAS2 in GD-OF. In contrast, HAS2 and UGDH expression dominate GD-OF and localize to CD34- OF. Recombinant human Slit2 (rhSlit2) substantially upregulates HA synthesis and HAS2 expression in fibrocytes but attenuates IL-6 and TNF-α production in these cells. In contrast, knocking down Slit2 in GD-OF reduces HA synthesis and HAS2 and UGDH expression while upregulating IL-6 and TNF-α. CONCLUSION: The dramatic differences in HA, IL-6, and TNF-α production, and HAS and UGDH expression found in fibrocytes and GD-OF appear, at least in part, to be attributable to Slit2. These findings provide novel insight into the differences in gene expression exhibited by CD34+ fibrocytes and CD34+ OF and therefore reveal important aspects of disease pathogenesis.

Integration of Sugar Metabolism and Proteoglycan Synthesis by UDP-glucose Dehydrogenase.

Regulation of proteoglycan and glycosaminoglycan synthesis is critical throughout development, and to maintain normal adult functions in wound healing and the immune system, among others. It has become increasingly clear that these processes are also under tight metabolic control and that availability of carbohydrate and amino acid metabolite precursors has a role in the control of proteoglycan and glycosaminoglycan turnover. The enzyme uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) produces UDP-glucuronate, an essential precursor for new glycosaminoglycan synthesis that is tightly controlled at multiple levels. Here, we review the cellular mechanisms that regulate UGDH expression, discuss the structural features of the enzyme, and use the structures to provide a context for recent studies that link post-translational modifications and allosteric modulators of UGDH to its function in downstream pathways.

Improved production of an acidic exopolysaccharide, the efficient flocculant, by Lipomyces starkeyi U9 overexpressing UDP-glucose dehydrogenase gene.

In order to increase content of glucuronic acid in the exopolysaccharide (EPS) and its flocculating activity, an UDP-glucose dehydrogenase gene was overexpressed in Lipomyces starkeyi V19. The obtained U9 strain could produce 62.1 ± 1.2 g/l EPS while the V19 strain only produced 53.5 ± 1.3 g/l EPS. The compositions of monosaccharides (mannose, glucuronic acid and galactose) in the purified EPS (U9-EPS) from the U9 strain contained 3.79:1:5.52 while those in the purified EPS (V19-EPS) were 3.94:1:6.29. The flocculation rate of the U9-EPS on kaolin clay reached 87.9%, which was significantly higher than that (74.7%) of the V19-EPS while the decolorization rate of Congo Red (CR) by the U9-EPS reached 94.3%, which was significantly higher than that of CR by the V19-EPS (86.23%). The results showed that the purified bioflocculant U9-EPS had effective flocculation of kaolin clay. The U9-EPS also had high ability to flocculate the polluted river water and decolorize Congo red.

Targeting UDP-glucose dehydrogenase inhibits ovarian cancer growth and metastasis.

More than 70% of patients with ovarian cancer are diagnosed in advanced stages. Therefore, it is urgent to identify a promising prognostic marker and understand the mechanism of ovarian cancer metastasis development. By using proteomics approaches, we found that UDP-glucose dehydrogenase (UGDH) was up-regulated in highly metastatic ovarian cancer TOV21G cells, characterized by high invasiveness (TOV21GHI ), in comparison to its parental control. Previous reports demonstrated that UGDH is involved in cell migration, but its specific role in cancer metastasis remains unclear. By performing immunohistochemical staining with tissue microarray, we found overexpression of UGDH in ovarian cancer tissue, but not in normal adjacent tissue. Silencing using RNA interference (RNAi) was utilized to knockdown UGDH, which resulted in a significant decrease in metastatic ability in transwell migration, transwell invasion and wound healing assays. The knockdown of UGDH caused cell cycle arrest in the G0 /G1 phase and induced a massive decrease of tumour formation rate in vivo. Our data showed that UGDH-depletion led to the down-regulation of epithelial-mesenchymal transition (EMT)-related markers as well as MMP2, and inactivation of the ERK/MAPK pathway. In conclusion, we found that the up-regulation of UGDH is related to ovarian cancer metastasis and the deficiency of UGDH leads to the decrease of cell migration, cell invasion, wound healing and cell proliferation ability. Our findings reveal that UGDH can serve as a prognostic marker and that the inhibition of UGDH is a promising strategy for ovarian cancer treatment.

GR/Sp3/HDAC1/UGDH signaling participated in the maternal dexamethasone-induced dysplasia of the rat fetal growth plate.

Maternal dexamethasone decreases the body length of the newborn. However, whether dexamethasone inhibits the development of the growth plate of the fetal long bone is still unknown. Here, we found that lengths of fetal femur and growth plate were both shorter in the fetuses with maternal dexamethasone (0.2 mg/kg.d from gestation day 9 to 20), with a decreased proteoglycan content of the growth plate in the fetal rat. Notable decreases in both the gene expression and H3K9 acetylation of UDP-glucose dehydrogenase (Ugdh) gene, which codes a key enzyme in the proteoglycan biosynthesis in the chondrocyte, were also observed. Meanwhile, up-regulation of glucocorticoid receptor (GR), specific protein 3 (Sp3), and histone deacetylase 1 (Hdac1) gene expression were detected in the fetal growth plate. Similar changes were also observed in the chondrogenic rat bone marrow stromal cells (BMSCs) with excessive exogenous dexamethasone. However, antagonizing GR with RU486 and silencing Hdac1 or Sp3 with specific siRNAs could all stimulate the H3K9 acetylation and gene expression of Ugdh previously inhibited by dexamethasone. Meanwhile, dexamethasone also induced the nuclear translocation of GR, which further directly bound to the Ugdh promoter and interacted with HDAC1 and Sp3, respectively. Collectively, our study revealed that maternal dexamethasone induced the direct binding of GR to the Ugdh promoter of the chondrocyte in the rat fetal growth plate, which recruited HDAC1 and Sp3, induced deacetylation of the H3K9, and subsequently inhibited Ugdh gene expression. Such changes further led to attenuated proteoglycan synthesis in the developing chondrocyte and therefore disrupted the development of growth plate and fetal long bone.

UDP-glucose 6-dehydrogenase regulates hyaluronic acid production and promotes breast cancer progression.

An improved understanding of the biochemical alterations that accompany tumor progression and metastasis is necessary to inform the next generation of diagnostic tools and targeted therapies. Metabolic reprogramming is known to occur during the epithelial-mesenchymal transition (EMT), a process that promotes metastasis. Here, we identify metabolic enzymes involved in extracellular matrix remodeling that are upregulated during EMT and are highly expressed in patients with aggressive mesenchymal-like breast cancer. Activation of EMT significantly increases production of hyaluronic acid, which is enabled by the reprogramming of glucose metabolism. Using genetic and pharmacological approaches, we show that depletion of the hyaluronic acid precursor UDP-glucuronic acid is sufficient to inhibit several mesenchymal-like properties including cellular invasion and colony formation in vitro, as well as tumor growth and metastasis in vivo. We found that depletion of UDP-glucuronic acid altered the expression of PPAR-gamma target genes and increased PPAR-gamma DNA-binding activity. Taken together, our findings indicate that the disruption of EMT-induced metabolic reprogramming affects hyaluronic acid production, as well as associated extracellular matrix remodeling and represents pharmacologically actionable target for the inhibition of aggressive mesenchymal-like breast cancer progression.

UDP-glucose accelerates SNAI1 mRNA decay and impairs lung cancer metastasis.

Cancer metastasis is the primary cause of morbidity and mortality, and accounts for up to 95% of cancer-related deaths1. Cancer cells often reprogram their metabolism to efficiently support cell proliferation and survival2,3. However, whether and how those metabolic alterations contribute to the migration of tumour cells remain largely unknown. UDP-glucose 6-dehydrogenase (UGDH) is a key enzyme in the uronic acid pathway, and converts UDP-glucose to UDP-glucuronic acid4. Here we show that, after activation of EGFR, UGDH is phosphorylated at tyrosine 473 in human lung cancer cells. Phosphorylated UGDH interacts with Hu antigen R (HuR) and converts UDP-glucose to UDP-glucuronic acid, which attenuates the UDP-glucose-mediated inhibition of the association of HuR with SNAI1 mRNA and therefore enhances the stability of SNAI1 mRNA. Increased production of SNAIL initiates the epithelial-mesenchymal transition, thus promoting the migration of tumour cells and lung cancer metastasis. In addition, phosphorylation of UGDH at tyrosine 473 correlates with metastatic recurrence and poor prognosis of patients with lung cancer. Our findings reveal a tumour-suppressive role of UDP-glucose in lung cancer metastasis and uncover a mechanism by which UGDH promotes tumour metastasis by increasing the stability of SNAI1 mRNA.

Catalytic mechanism of UDP-glucose dehydrogenase.

UDP-glucose dehydrogenase (UGDH), an oxidoreductase, catalyzes the NAD+-dependent four-electron oxidation of UDP-glucose to UDP-glucuronic acid. The catalytic mechanism of UGDH remains controversial despite extensive investigation and is classified into two types according to whether an aldehyde intermediate is generated in the first oxidation step. The first type, which involves the presence of this putative aldehyde, is inconsistent with some experimental findings. In contrast, the second type, which indicates that the first oxidation step bypasses the aldehyde via an NAD+-dependent bimolecular nucleophilic substitution (SN2) reaction, is consistent with the experimental phenomena, including those that cannot be explained by the first type. This NAD+-dependent SN2 mechanism is thus more reasonable and likely applicable to other oxidoreductases that catalyze four-electron oxidation reactions.

Clinicopathological and prognostic significance of nuclear UGDH localization in lung adenocarcinoma.

This study aimed to clarify relationships among UDP-glucose-6 dehydrogenase (UGDH) expression, clinicopathological factors, and the prognosis of patients, and to determine the role of UGDH in lung adenocarcinoma (AC). Firstly, UGDH expression and localization in 126 lung AC tissues were immunohistochemically studied, and associations with clinicopathological parameters and patients' prognosis were evaluated. Secondly, serum UGDH levels were measured in 267 lung cancer patients and 100 healthy controls. Finally, the effects of UGDH knockdown by siRNA on migration and invasion abilities were analyzed. As a result, nuclear UGDH staining was significantly correlated with poorer differentiation, a larger tumor size, higher p-TNM stage, positive nodal metastasis, positive lymphatic invasion, and positive vascular invasion in lung AC patients. Nuclear UGDH-positive patients showed significantly poorer survival than nuclear UGDH-negative patients. Serum UGDH levels were especially higher in lung AC patients even in stage I than those in healthy controls. In lung AC cell lines, nuclear expression levels of UGDH were higher in LC-2/ad cells than in A549 cells. UGDH siRNA-treated LC-2/ad cells showed significantly decreased migration and invasion abilities, but no significant differences were observed in UGDH siRNA-treated A549 cells. These data indicate that UGDH expression and localization are an early sero-diagnostic marker in addition to a poor prognostic indicator in lung AC patients.

UDP-glucose Dehydrogenase: The First-step Oxidation Is an NAD+-dependent Bimolecular Nucleophilic Substitution Reaction (SN2).

UDP-glucose dehydrogenase (UGDH) catalyzes the conversion of UDP-glucose to UDP-glucuronic acid by NAD+-dependent two-fold oxidation. Despite extensive investigation into the catalytic mechanism of UGDH, the previously proposed mechanisms regarding the first-step oxidation are somewhat controversial and inconsistent with some biochemical evidence, which instead supports a mechanism involving an NAD+-dependent bimolecular nucleophilic substitution (SN2) reaction. To verify this speculation, the essential Cys residue of Streptococcus zooepidemicus UGDH (SzUGDH) was changed to an Ala residue, and the resulting Cys260Ala mutant and SzUGDH were then co-expressed in vivo via a single-crossover homologous recombination method. Contrary to the previously proposed mechanisms, which predict the formation of the capsular polysaccharide hyaluronan, the resulting strain instead produced an amide derivative of hyaluronan, as validated via proteinase K digestion, ninhydrin reaction, FT-IR and NMR. This result is compatible with the NAD+-dependent SN2 mechanism.

Tubby-like Protein 2 regulates homogalacturonan biosynthesis in Arabidopsis seed coat mucilage.

KEY MESSAGE: A possible transcription factor TLP2 was identified to be involved in the regulation of HG biosynthesis in Arabidopsis seed mucilage. TLP2 can translocate into nucleus from plasma membrane by interacting with NF-YC3. The discovery of TLP2 gene function can further fulfill the regulatory network of pectin biosynthesis in Arabidopsis thaliana. Arabidopsis seed coat mucilage is an excellent model system to study the biosynthesis, function and regulation of pectin. Rhamnogalacturonan I (RG-I) and homogalacturonan (HG) are the major polysaccharides constituent of the Arabidopsis seed coat mucilage. Here, we identified a Tubby-like gene, Tubby-like protein 2 (TLP2), which was up-regulated in developing siliques when mucilage began to be produced. Ruthenium red (RR) staining of the seeds showed defective mucilage of tlp2-1 mutant after vigorous shaking compared to wild type (WT). Monosaccharide composition analysis revealed that the amount of total sugars and galacturonic acid (GalA) decreased significantly in the adherent mucilage (AM) of tlp2-1 mutant. Immunolabelling and dot immunoblotting analysis showed that unesterified HG decreased in the tlp2-1 mutant. Furthermore, TLP2 can translocate into nucleus by interacting with Nuclear Factor Y subunit C3 (NF-YC3) to function as a transcription factor. RNA-sequence and transactivation assays revealed that TLP2 could activate UDP-glucose 4-epimerase 1 (UGE1). In all, it is concluded that TLP2 could regulate the biosynthesis of HG possibly through the positive activation of UGE1.

Hysteresis and Allostery in Human UDP-Glucose Dehydrogenase Require a Flexible Protein Core.

Human UDP-glucose dehydrogenase (hUGDH) oxidizes UDP-glucose to UDP-glucuronic acid, an essential substrate in the phase II metabolism of drugs. The activity of hUGDH is regulated by the conformation of a buried allosteric switch (T131 loop/α6 helix). Substrate binding induces the allosteric switch to slowly isomerize from an inactive E* conformation to the active E state, which can be observed as enzyme hysteresis. When the feedback inhibitor UDP-xylose binds, the allosteric switch and surrounding residues in the protein core repack, converting the hexamer into an inactive, horseshoe-shaped complex (EΩ). This allosteric transition is facilitated by large cavities and declivities in the protein core that provide the space required to accommodate the alternate packing arrangements. Here, we have used the A104L substitution to fill a cavity in the E state and sterically prevent repacking of the core into the EΩ state. Steady state analysis shows that hUGDHA104L binds UDP-xylose with lower affinity and that the inhibition is no longer cooperative. This means that the allosteric transition to the high-UDP-xylose affinity EΩ state is blocked by the substitution. The crystal structures of hUGDHA104L show that the allosteric switch still adopts the E and E* states, albeit with a more rigid protein core. However, the progress curves of hUGDHA104L do not show hysteresis, which suggests that the E* and E states are now in rapid equilibrium. Our data suggest that hysteresis in native hUGDH originates from the conformational entropy of the E* state protein core.

The entropic force generated by intrinsically disordered segments tunes protein function.

Protein structures are dynamic and can explore a large conformational landscape1,2. Only some of these structural substates are important for protein function (such as ligand binding, catalysis and regulation)3-5. How evolution shapes the structural ensemble to optimize a specific function is poorly understood3,4. One of the constraints on the evolution of proteins is the stability of the folded 'native' state. Despite this, 44% of the human proteome contains intrinsically disordered peptide segments greater than 30 residues in length6, the majority of which have no known function7-9. Here we show that the entropic force produced by an intrinsically disordered carboxy terminus (ID-tail) shifts the conformational ensemble of human UDP-α-D-glucose-6-dehydrogenase (UGDH) towards a substate with a high affinity for an allosteric inhibitor. The function of the ID-tail does not depend on its sequence or chemical composition. Instead, the affinity enhancement can be accurately predicted based on the length of the intrinsically disordered segment, and is consistent with the entropic force generated by an unstructured peptide attached to the protein surface10-13. Our data show that the unfolded state of the ID-tail rectifies the dynamics and structure of UGDH to favour inhibitor binding. Because this entropic rectifier does not have any sequence or structural constraints, it is an easily acquired adaptation. This model implies that evolution selects for disordered segments to tune the energy landscape of proteins, which may explain the persistence of intrinsic disorder in the proteome.